Concerns about environmental problems and limited resource depletion have increased rapidly on a world-wide basis in recent years. Accordingly, there is an increasing interest in constructing an environmentally-friendly and reproducible organism-based production system as an alternative to these concerns. The system is capable of being constructed by regulating a metabolic pathway in an organism to optimize metabolic flow for production of desired metabolites, wherein various molecular biology techniques are required in this process. To regulate the metabolic pathway, there are two major techniques including one technology of increasing an enzyme expression required for a biosynthesis process to enhance metabolism flow required for the production of metabolites, and the other technology of blocking the metabolic flow used for cell growth and other metabolite productions and of deleting genes for utilization as target metabolite production. The currently widely used gene knock-out method is to substitute a gene to be deleted with any sequences having homologous sequences through recombinase, thereby losing function thereof (Datsenko et al, PNAS, 97(12): 6640-6645, 2000). However, this gene knock-out method has various problems.
First of all, the gene knock-out method requires several weeks—several months, and the gene expression is able to be regulated only by on-off, and gene deletion needs to be repeated for each strain to be applied to several strains. Therefore, it is the most time-consuming process in metabolic engineering studies in which a number of cases have to be confirmed experimentally. A method of inducing mutations in a promoter of a gene present on chromosome to weaken the activity or a method of substituting with various promoters that are capable of regulating gene expression instead of the existing promoters are used as a method for regulating and inhibiting a gene expression level. However, these methods are also performed through the same or similar processes as the gene knock-out method, and thus, they have limitation of the gene knock-out method through chromosomal recombination, and further, unlike the gene knock-out method, it is difficult to predict results until an expression level resulted from the mutant promoter is directly identified. Consequently, these methods may require more effort and time than simple gene knock-out method.
In order to overcome the limitations of this conventional gene inhibition method, a short-length customized synthetic sRNA technique has been developed (Na, D et al., Nat. Biotechnol., 31(2), 170-174, 2013; Yoo, S M et al. Nat. Protoc., 8(9), 1694-1707, 2013), which have advantages in that it is possible to easily reduce an expression of a target gene by using a plasmid without modifying chromosomal sequence of a target strain, and to easily apply the same gene expression inhibition to various strains in a multiple simultaneous manner. Moreover, this technique is quick and simple in view of construction, storage, and application.
An sRNA consists of Hfq binding sequence and a base pairing region (hereinafter, referred to as BPR) for complementary binding to the mRNA of the target gene. In order to regulate expression of the target gene, in the conventional technique, the binding free energy of the BPR part is calculated to control a base sequence length and mutation. This is advantageous because the BPR part is capable of being designed based on the base sequence information of the target gene and synthetic sRNA is capable of being easily designed and constructed through general gene recombination method. However, in order to fine-tune gene expression to a desired level, the BPR part of the sRNA needs to be redesigned for each target gene and the sRNA needs to be redesigned based on the redesigned BPR part. Further, the strategy using the binding free energy has a limitation in that it is difficult to accurately predict changes in sRNA activity. The binding free energy is calculated based on the sequence of sRNA and mRNA. In an actual binding of sRNA and mRNA, a prediction accuracy may be lowered because Hfq protein helps the binding of sRNA and mRNA. Further, when structural changes occur in the binding sequence due to a secondary structure of sRNA and mRNA, it is not possible to predict the degree of expression inhibitory activity. To overcome these problems, a general-purpose system capable of regulating the gene expression to a desired level regardless of the type of target gene or the sequence of BPR should be developed.
Therefore, the present inventors made an effort to construct the general-purpose gene expression regulatory system that meets the above conditions, and as a result, attempted mutation of other elements while fixing the BPR, and consequently, the general-purpose gene expression regulatory system was constructed by introducing promoters having various strength activities and applying a mutation to a Hfq binding site in the sRNA scaffold. It was confirmed that the expression of the target gene could be regulated by introducing various constitutive promoters to regulate the expression level of sRNA, and when an inducible promoter was introduced, it was confirmed that the expression of the target gene could be fine-tuned by introducing a mutation into the Hfq binding site of the sRNA scaffold, thereby completing the present invention.